ABSTRACT As ?omics? studies become increasing common, new methods to identify biomolecule interactions are needed. The ability to isolate specific biomolecules is a fundamental process that spans many biomedical sectors including research, diagnostics, drug discovery, and biomarker development. Commonly, such isolations are performed by capturing the analyte of interest on a solid structure and then washing away unbound or non- interacting material. This washing-based strategy has important limitations for interactions that are transiently or weakly captured (e.g., protein complexes involved in post-translational modification). Protein-protein interactions govern cellular behavior and are consequently transient in nature. Once captured, dilutive washing further promotes complex dissociation, making it difficult to isolate important transient complexes, including transcriptional machinery and enzyme/substrate interactions. Yet, it is these dynamic regulatory complexes that are appealing as drug targets, due to the ability to directly modulate cell behavior through their disruption. Improved ability to isolate and analyze weakly bound analytes would be transformative across biomedical disciplines, enabling scientists to ?see? what was previously unknown. We will apply a fundamentally new isolation process, Exclusion-based Sample Preparation (ESP), to this important category of analytes. ESP relies on the passage of magnet bead-bound analyte through phase interfaces to achieve purification with much less exposure to pro-dissociation factors such as dilution. Here, using a novel model system with ?tunable? affinity, we will perform a comprehensive characterization of the ESP advantage to drive widespread acceptance and commercialization of this method. Additionally, we will apply ESP to the capture of weakly bound analytes in animal model tissue samples for the first time, validating ESP's utility with ?real world? samples. We will isolate protein complexes that are implicated in the ubiquitous NF-kB pathway, using splenic samples from mice with specific NF-kB modulating mutations. Successful completion of both Aims will provide a comprehensive quantitative characterization of the technique as well as demonstrating its ?discovery? capabilities ? together these will lay the foundation for immediate marketing of ESP into this market as well as future (Phase II) ?discovery-based? studies aimed at mapping unknown branches of the ?interactome?, with the ultimate goal of discovering new drug targets to benefit human health. !